Materials and methods for detection of enterovirus and norovirus

ABSTRACT

The invention provides polynucleotides and methods for detecting and quantifying RNA viruses, such as enteroviruses and noroviruses. In one aspect, the invention provides amplification primers and labeled molecular beacons for amplification of viral nucleic acid sequences. In another aspect, the invention provides a synthetic RNA internal control. In another aspect, the invention provides a kit for detecting the presence of enterovirus and/or norovirus in a sample.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.10/938,005, filed Sep. 10, 2004, which is a continuation-in-part of U.S.application Ser. No. 10/857,109, filed May 28, 2004, which is acontinuation-in-part of U.S. application Ser. No. 10/179,082, filed Jun.25, 2002, which claims the benefit of U.S. Provisional Application No.60/301,218, filed Jun. 27, 2001, each of which are incorporated hereinby reference in their entireties, including all figures, tables, andsequences.

GOVERNMENT SUPPORT

The subject matter of this application has been supported by a researchgrant from the Office of Naval Research under grant numberN0014-03-1-0753. Accordingly, the government may have certain rights inthis invention.

BACKGROUND OF THE INVENTION

Good microbiological water quality in coastal waters is a nationalpriority. With wastewater contaminating such aquatic areas, there is anincreased risk of infection. The organisms responsible for infectiousrisk can include viruses, bacteria, and protozoans. Individuals at riskare those who have increased contact with contaminated water. Theseindividuals nonexclusively include swimmers, divers, and boaters as wellas those consuming products harvested from the contaminated water.

Pathogenic organisms such as enteric viruses pose a serious problem tolife. Of the enteric viruses, one of the largest and best known groupsis that of the enteroviruses, some of them important pathogens forhumans and animals. Enteroviruses (EV) are small, non-enveloped, andisometric, belonging to the Picornaviridae family, and their genomeconsists of a simple chain RNA positive pole molecule (ssRNA+) withapproximately 7,400 nucleotides (nt). These are the among the mostcommon animal viruses and infect a large variety of mammals. The virusesare generally transmitted from person to person by ingestion (forexample, fecal-oral route) or from exposure to contaminated watersupplies. Furthermore, evidence indicates that the viruses can be spreadvia the respiratory tract. Once exposed, the virus infects the body viathe blood stream and multiplies in the gut mucosa.

Approximately 90 serotypes of enteroviruses have been identified, 62 ofwhich infect humans and 27 animals. Bovine enteroviruses (BEV) arebroadly distributed all over the world and classified as enzootic insome countries. Two species have been described to date: BEV1 and BEV2(Knowles, N. J., and Barnett, I. T. (1985), Arch Virol 83 (3-4),141-55), although there are probably more that have not yet beenidentified. There are 3 species of porcine enteroviruses that have beendescribed. An ovine enterovirus (OEV-1) that is closely linked to theBEVs has been isolated. The existence of viruses has been described inocean environments, although it is believed that most of the virusesfound in natural water are bacteriophages, cyanophages, and other typescapable of infecting microalgae. The existence of water contaminated byhuman enteric viruses has also been described, especially close to urbanareas, related to sources of human infections (Chapron et al., 2000,Appl. Environ. Microbiol. 66:2520-2525; Abbaszadegan et al., 1993, Appl.Environ. Microbiol. 59:1318-1324; Abbaszadegan et al., 1999, Appl.Environ. Microbiol. 65:444-449; Bosch A., 1998, Int. Microbiol.1:191-196; Pianetti et al., 2000, Epidemiol. Infect. 125:455-462;Schvoerer et al., Res. Microbiol. 152:179-186).

Most infections occur during childhood. Although the infections arelargely transient, they produce lifelong immunity to the organism. Amajority of enteroviral infections result in mild illness; however,enteroviruses can cause an array of different diseases affecting manydifferent organs (for example, neurologic (polio, aseptic meningitis,encephalitis), respiratory (common cold, tonsillitis, pharyngitis,rhinitis), cardiovascular (myocarditis, pericarditis), etc.). Theability to detect the presence of the organisms that cause theseconditions is beneficial to the health and welfare of those withincreased potential of exposure.

There is no specific treatment for enterovirus infections. In infants,enteroviral meningitis is often confused with bacterial or herpesvirusinfections resulting in misdiagnosis. Often, children are hospitalizedand incorrectly treated with antibiotics and anti-herpes drugs.

While most enteroviral infections are known and documented, theseviruses have also been implicated in several chronic diseases, such asjuvenile onset of diabetes mellitus, chronic fatigue syndrome, andamyotrophic lateral sclerosis (Lou Gehrig's Disease); however,definitive proof is deficient.

Furthermore, there is a high degree of serological cross reactivityamongst the more than 70 known enteroviruses, which include: Polio 1, 2,3; Coxsackie A 1-24; Coxsackie B 1-6; ECHO 1-34; Entero 68-71; andEntero 72 (Hepatitis A). To reduce the risk of enteroviral infection,the U.S. EPA proposed legislation mandating the testing of groundwaterfor the presence of enteroviruses.

Noroviruses (genus Norovirus, family Caliciviridae) are a group ofrelated, single-stranded RNA, non-enveloped viruses that cause acutegastroenteritis in humans. Norovirus was recently approved as theofficial genus name for the group of viruses provisionally described as“Norwalk-like viruses” (NLV). This group of viruses has also beenreferred to as caliciviruses (because of their virus family name) and assmall round structured viruses, or SRSVs (because of their morphologicalfeatures). Another genus of the calicivirus family that can causegastroenteritis in humans is Sapovirus, formerly described as“Sapporo-like virus” (SLV) and sometimes referred to as classical ortypical calicivirus.

Noroviruses are named after the original strain “Norwalk virus,” whichcaused an outbreak of gastroenteritis in a school in Norwalk, Ohio, in1968. Currently, there are at least four norovirus genogroups (GI, GII,GIII, and GIV), which in turn are divided into at least 20 geneticclusters.

Noroviruses are transmitted primarily through the fecal-oral route,either by consumption of fecally contaminated food or water or by directperson-to-person contact. Environmental and fomite contamination mayalso act as a source of infection. Evidence exists for transmission dueto aerosolization of vomitus that presumably results in dropletscontaminating surfaces or entering the oral mucosa and being swallowed.Noroviruses are highly contagious, and it is thought that an inoculumbearing very few viral particles is sufficient to infect an individual.During outbreaks of norovirus gastroenteritis, several modes oftransmission have been documented; for example, initial foodbometransmission in a restaurant, followed by secondary person-to-persontransmission to household contacts. Although presymptomatic viralshedding may occur, shedding usually begins with onset of symptoms andmay continue for 2 weeks after recovery.

The isolation and detection of such enteric pathogens is known to beaccomplished by reverse transcriptase polymerase chain reaction (RT-PCR)and cell culture (See M. Gilgen et al., (1995) Appl. Environ. Microbiol.61: 1226-31). Both of these techniques have advantages anddisadvantages. While RT-PCR is generally faster, more sensitive, andmore specific, it cannot distinguish viable from nonviable viruses.Further, currently available primer sets are not specific among the fullsuite of human enteroviruses. Currently only about 25 of the more than70 known enteroviruses can be detected by the RT-PCR method. Thus, useof the RT-PCR assay allows sensitive detection but cannot determinewhether the resulting amplicon is from one virus, multiple virusstrains, a pathogen, or a vaccine.

BRIEF SUMMARY OF THE INVENTION

The instant invention provides an amplification and detection technologyfor enteroviruses and noroviruses in samples, such as biological andenvironmental samples, based upon an enzymatic amplification reaction,such as nucleic acid sequence based amplification (NASBA) or RT-PCR.NASBA is an isothermal method of amplifying RNA (Compton, J. Nature,1991, 350(6313):91-92). The process results in an approximatebillion-fold amplification of the RNA target in less than two hours anddoes not utilize Taq polymerase or thermal cycling. It has beendescribed as a self-sustained sequence reaction (3 SR; Guatelli et al.,Proc. Natl. Acad. Sci. USA, 1990, 87(5):1874-1878) andtranscription-based amplification (TAS; Kwoh, D. Y. et al., Proc. Natl.Acad. Sci. USA, 1989, 86(4): 1173-1177).

Key components of NASBA are the conversion of RNA into DNA by the actionof reverse transcriptase and the production of RNA by T7 RNA polymerase.First, in the non-cyclic or linear phase of the reaction, a primer (PI)is bound at the 3′ end of messenger RNA (mRNA). This primer is unusualin that it contains a T7 RNA polymerase promoter. Second, AMV reversetranscriptase converts the molecule to a RNA/DNA hybrid. RNAse Hspecifically degrades the RNA in the hybrid and the AMV reversetranscriptase converts the single stranded DNA into double stranded DNA.Finally, T7 RNA polymerase recognizes the T7 RNA polymerase promoter,initiating the cyclic phase. Antisense RNA product is produced and theAMV reverse transcript makes a DNA/RNA hybrid. RNAse H degrades the RNA,a duplex DNA molecule is synthesized, T7 RNA polymerase makes RNA, andthe cycle continues.

The method combines current sample concentration and NASBA technologywith novel nucleotide primers to amplify the viral RNA. Samples areobtained and can be concentrated by any method known in the art (forexample, charged filters, filterite cartridges, vortex flow filtration,etc.) or, alternatively, left unconcentrated. The viral RNA is extractedby a combination of heating and Rneasy extraction. Utilizing novelenteroviral or noroviral primers, the RNA is amplified using NASBAtechnology. The RNA is then detected using a method known in the art(for example, by gel electrophoresis, molecular probing, or fluorescentmolecular beacons).

In another aspect, the present invention provides primers, probes,molecular beacons, specific internal controls, and methods for detectionand quantitation of noroviruses and enteroviruses in samples, such asbiological or environmental samples. These methods (assays) are basedupon the amplification of a portion of the viral RNA polymerase gene,using a method such as NASBA for amplification, and detection using alabeled (e.g., fluorescently labeled) molecular beacon. The norovirusassay of the invention can detect viruses within the genogroup II familyof noroviruses, and can detect 0.1 fg. noroviral RNA (approximately 10³virus particles). The enterovirus assay can routinely detectapproximately 10 enteroviral particles. In one embodiment, the sample isan environmental sample, such as soil, food, beverages, feed, water(e.g., fresh water, salt water, waste water, drinking water), sewage,sludge, and surfaces or samples obtained from surface swipes. In anotherembodiment, the sample is a biological specimen, such as a clinicalsample (e.g., stool, saliva, blood, urine, cerebrospinal fluid, etc.).In another embodiment, the sample comprises an organism or tissue froman organism, such as shellfish, which are potential hosts fornoroviruses and enteroviruses.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a gel electrophoresis illustrating the effects of NASBAamplification of enteroviral genomes.

FIGS. 2A and 2B show, respectively, an amplification plot and standardcurve of enteroviruses based upon viral direct counts.

FIG. 3 shows the standard curve of a norovirus transcript. Eachconcentration is in duplicate from 100 fg to 0.1 fg.

FIG. 4 shows a schematic of an internal control for enterovirusIC-NASBA, according to the subject invention.

FIG. 5 shows the results of a typical IC-real time NASBA reactioncontaining 10,000 internal control copies. Dashed lines indicate thethreshold cycle (Ct) for each of the wild-type and internal control.

FIGS. 6A and 6B show standard curves of enteroviral RNA signals plottedagainst the Ct (FIG. 6A) and against the Ct ratio (FIG. 6B). A muchbetter relationship is apparent in the Ct ratio method (R2=0.9882 for Ctratio [IC-NASBA] vs. 0.7379 for Ct value method.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO:1 is the nucleotide sequence for primer Ent P1 (=JP 127).

SEQ ID NO:2 is the nucleotide sequence for primer Ent P2 (=JP128).

SEQ ID NO:3 is the nucleotide sequence for a probe specific fordetecting poliovirus.

SEQ ID NO:4 is the nucleotide sequence for primer Ent P3.

SEQ ID NO:5 is the nucleotide sequence for EnteroBeacon.

SEQ ID NO:6 is the nucleotide sequence for primer Nor3.

SEQ ID NO:7 is the nucleotide sequence for primer Nor4.

SEQ ID NO:8 is the nucleotide sequence for NorBeacon.

SEQ ID NO:9 is the nucleotide sequence for the Enteroviral InternalControl.

SEQ ID NO:10 is the nucleotide sequence for the beacon for theenteroviral internal control.

DETAILED DISCLOSURE OF THE INVENTION

The subject invention provides polynucleotides and methods for detectingthe presence of an RNA virus, such as enterovirus and norovirus, withina sample. The method of the invention can include a series ofbiochemical steps preparatory to commencing the detection assay. In oneembodiment, the invention provides an isolated polynucleotide comprisinga nucleic acid sequence selected from the group consisting of SEQ IDNOs:1-10, or a fragment thereof. In another embodiment, the inventionprovides an isolated polynucleotide consisting of a nucleic acidsequence selected from the group consisting of SEQ ID NOs:1-10, or afragment thereof.

The method of the invention includes amplifying the viral RNA from thesample using an isolated polynucleotide (amplification primer and/orbeacon) of the invention, which is capable of selectively hybridizingwith the viral nucleic acids. Preferably, the polynucleotides areselected from the group consisting of SEQ ID NO:1, SEQ ID NO:4, and SEQID NO:5 (primer Ent P1, primer Ent P3, and EnteroBeacon, respectively),for detection of enterovirus. Preferably, the polynucleotides areselected from the group consisting of SEQ ID NO:6, SEQ ID NO:7, and SEQID NO:8 (primer Nor3, primer Nor4, and NorBeacon, respectively), fordetection of norovirus.

Samples are obtained and are tested, either unconcentrated or afterconcentration by a method known in the art (for example, chargedfilters, filterite cartridges, vortex flow filtration, etc). Currentsample concentration and NASBA technology is combined with novelnucleotide primers to amplify the viral RNA contained therein. The viralRNA can be extracted by a combination of heating and Rneasy extraction.Utilizing novel enteroviral and/or norovirus primers, the RNA isamplified using NASBA technology or other enzyme-based amplificationreaction.

The method of the subject invention involves obtaining or providing asample (also referred to herein as the test sample). The test sample canbe taken from anywhere susceptible to contain viruses such asenterovirus and norovirus, for which there is a desire to detect theviral presence. For instance, such a test sample can be taken from ahuman or non-human subject, such as a companion animal (such as a dog orcat), drinking water, or livestock (e.g., bovine, porcine, etc.)facilities, livestock drinking water, the surrounding environment(pastures, water, etc.), from environmental specimens, from aquaticmedia in direct or indirect contact with the source of contamination,for example, effluents of the livestock facility containing livestockwaste, rivers close to the source of contamination or crossing thepossible source of contamination, food for humans, food for companionanimals, food for livestock, etc. The source of contamination may be,for example, a livestock facility, typically a bovine or porcinelivestock farm. The test sample can be surface matter that may containor otherwise support the presence of the virus.

The test sample can be any sample susceptible to containing an entericvirus, such as enteroviruses and noroviruses. For example, the testsample can be a biological sample, such as blood, saliva, urine, sputum,nasal mucus, feces (stool), host cells, host tissues, tissuehomogenates, muscle, liver, thymus, cerebrospinal fluid, etc.; or anenvironmental sample such as an environmental specimen, a sample ofpasture, a water sample, a sample of fish or molluscs found inpotentially contaminated water, clothing, eating utensils, etc. The testsamples can be subjected to purifying protocols known in the art or usedin the detection analysis directly. For example, column chromatography,density centrifugation, or ammonium sulfate precipitation. These andother methods are disclosed, for example, in Sambrook et al., MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York(1989). For simplicity and ease of assay, it is preferred that thesample be used directly without purification.

The test sample is then subjected to an appropriate treatment to removeor extract the nucleic acid contained in the viral particles eventuallypresent in the test sample. The RNA can be extracted by conventionalmethods, using conventional kits and reagents and then placed in contactwith the reagents for amplification, e.g., NASBA, the reversetranscription (RT) reaction and for the polymerase chain reaction (PCR)in conditions in which a sequence of target nucleotides present in thegenome of the enteric virus with an RNA genome is amplified to form anamplification product. The RT-PCR reaction can be performed usingconventional methods, either in a single stage or in two stages. Thoseskilled in the art are familiar with both alternatives.

As used herein, the term “primer” means an isolated or purifiedpolynucleotide which is produced synthetically or biologically andincludes a specific nucleotide sequence which permits hybridization to asection containing the target nucleotide sequence. Definedprimers/polynucleotides may be produced by any of several well knownmethods, including automated solid-phase chemical synthesis usingcyanoethylphosphoramidite precursors. Other well-known methods forconstruction of synthetic primers/oligonucleotides may, of course, beemployed (J. Sambrook, E. F. Fritsch and T. Maniatis, Molecular Cloning11 (2d ed. 1989).

The primers and molecular beacons used to amplify the sample nucleicacids (e.g., SEQ ID NOs:1-10, or functional fragments thereof) may becoupled to a detectable moiety (a label). A preferred example of such adetectable moiety is fluorescein, which is a standard label used innucleic acid sequencing systems using laser light as a detection system.Other detectable labels can also be employed to label the primers,molecular beacons, and probes of the invention, however, including otherfluorophores, radio labels, chemical couplers such as biotin which canbe detected with streptavidin-linked enzymes, and epitope tags such asdigoxigenin detected using antibodies. The primers, probes, andmolecular beacons may be modified whereby another nucleotide is added toor substituted for at least one nucleotide in the oligonucleotide. Theterm “add(ed)” means that nucleotide, oligo dGTP, oligo DATP, oligodTTP, oligo dCTP, etc. having fluorescence substance, linker arm,biotin, etc. are bound to a 5′-terminal or a 3′-terminal of theoligonucleotide sequence. The term “substitute(d)” means that nucleotidehaving fluorescence substance, linker arm, biotin, etc. is introduced asa substitute for at least one nucleotide in the oligonucleotide.Introduction of known labels such as radioactive substances, enzymes,fluorescence substances, etc. after synthesis of the oligonucleotide isalso included therein.

The reagent mix to perform the RT of the RNA of an enteric viruscontains the reagents required for the reaction, for example water,dNTPs (dATP, dCTP, dGTP and dTTP), an appropriate buffer for the RTreaction, a reverse transcriptase, an initiator oligonucleotide (alsoknown as a primer) which blends with a region of the RNA of the entericvirus in question and then, due to the reverse transcriptase, allows aDNA complementary (cDNA) to the RNA to be obtained, etc. On the otherhand, the reagent mix for the PCR contains the reagents required for thereaction to take place, for example water, dNTPs, an appropriate bufferfor the PCR reaction, a heat-stable polymerase DNA, a pair of primers toamplify the target sequence of the genome of the enteric virus inquestion, a magnesium salt, etc. In specific embodiments, the entericvirus is a enterovirus or norovirus.

In another embodiment, the method of the invention is performed using asingle RT-PCR reagent mix containing the reagents required for thereaction to take place, for example water, dNTPs, an appropriate bufferfor the RT-PCR reactions, a reverse transcriptase, a heat-stablepolymerase DNA, etc. There are kits commercially available that providethis reaction mix, to which the appropriate primers are added. In thiscase, it is an advantage that the primers used for the RT reaction isone of the primers involved in the enzymatic amplification reaction(PCR).

To apply the method provided by this invention any primer that allowsone to obtain a cDNA of the enteric virus in question can be used, andany pair of primers that allows amplification of a target sequencewithin the viral RNA polymerase gene of the enteric virus in question.

Conventional techniques can be used for the RT-PCR reaction, inconditions in which an amplifiable DNA fragment, eventually present inthe reaction medium, representative of a target sequence of an entericvirus, is amplified to form an amplification product. The amplificationproduct is separated and analyzed using conventional techniques, forexample by electrophoresis, and sequenced. Next, the presence or absencein the test sample of an enteric virus containing the selected targetsequence is determined and the viral isolate is identified. Theidentification of the isolate of the enteric virus(es) detected can beby conventional methods, for example by sequencing the region of theamplified viral genome. The detection of an enteric virus in the testsample indicates the existence of environmental contamination orinfection.

Conventional techniques are used for the PCR, in conditions in which anamplifiable DNA fragment, eventually present in the reaction medium,representative of a target sequence of an enteric virus is amplified toform an amplification product. The amplification product is separatedand analyzed using any conventional method, for example byelectrophoresis, and sequenced, and the presence or absence of theenteric virus containing the selected target sequence is determined andthe viral isolate identified. The identification of the isolate of theenteric virus(es) detected can be by conventional methods, for exampleby sequencing the region of the amplified viral genome. The detection ofan enteric virus in the test sample can indicate the existence ofenvironmental contamination or infection, for example, depending uponthe nature and origin of the test sample. In a specific embodiment, theproducts of the RT-PCR reaction are separated by electrophoresis inagarose and viewed by conventional methods, for example ultravioletlight after staining with ethidium bromide.

The detection methods of the invention can be used in differentapplications, for instance to identify the existence of environmentalcontamination, or presence of infection, to discover the origin of thecontamination, to conduct epidemiological studies or studies related toviral molecular evolution, etc.

As it is used in this description, the expression “certain degree ofidentity” means that the identity between the isolates of the entericviruses detected and characterized in the test sample and the isolatesof the enteric viruses found in said possible source of contamination is50% or more, preferably 80% or more, and more preferably 95% or more,when the degree of identity is measured by molecular sequencing of theregion of the viral genome used in this study. If another less conservedregion of the genome of an enteric virus is used, these values could besomewhat different. Alternatively, other techniques may be used, forexample molecular typification, serological, etc., allowing one tocompare the isolates of enteric viruses and conclude that two isolatesof different enteric viruses belong to the same genotype or serotype.

The invention further includes a kit for the detection of enterovirusand/or norovirus, comprising at least a pair of primers allowing one toamplify a target sequence of the cDNA of the virus in question.

The kits provided by this invention can be in pack-form containing aplurality of recipients (e.g., compartments), including recipients withone or more primers, probes, and/or labeled molecular beacons of thesubject invention (e.g., SEQ ID NOs:1-10), recipients with all or partof the other reagents required for the method provided by thisinvention, for example water, dNTPs (dATP, dCTP, dGTP and dTTP), anappropriate buffer for the reaction (RT, PCR or RT-PCR), a reversetranscriptase, a heat-stable polymerase DNA, a magnesium salt, etc.Additionally and optionally, the kits provided by this invention caninclude recipients with nucleic acid (RNA, cDNA, DNA, etc.) fromnorovirus and/or enterovirus, well characterized for use as a positivecontrol. In a specific embodiment, the control is a specific internalcontrol of the subject invention, such as SEQ ID NO:9.

There are more than 70 known enteroviruses. In addition to the threedifferent polioviruses, there are 61 non-polio enteroviruses that cancause disease in humans: 29 coxsackieviruses (23 coxsackie A viruses and6 coxsackie B viruses, 28 echoviruses, and 4 other enteroviruses. Thepresence of each of these enteroviruses can be determined using thepolynucleotides and methods of the subject invention.

In a preferred embodiment, the Norovirus to be detected is a genotype IIstrain selected from the group consisting of GII-1 (Hawaii virus), GII-2(Melksham virus), GII-3 (Mexico virus), and GII-4 (Grimsby virus).

Optionally, the methods of the invention can be used to detect thepresence of enterovirus and norovirus in a sample simultaneously orconsecutively, in any order.

The terms “polynucleotide”, “oligonucleotide”, and “nucleic acidsequence” are used herein interchangeably to refer to nucleic acidsequences of any length. For example, DNA and/or RNA molecules arecomposed of various nucleotides and/or nucleotide analogs.

The term “fragments” as it relates to polynucleotides and nucleic acidmolecules (including primers) is understood to mean those fragmentswhich are specific for the particular enteric virus (e.g., enterovirusor norovirus) when used for methods to identify the presence of theenteric virus and specific for an enteric virus serotype when used in amethod for serotyping an enteric virus. For example, fragments of apolynucleotide of the subject invention (i.e., a primer, probe,molecular beacon, internal control) can be up to one nucleotide lessthan the particular full length sequence. For example, fragments cancomprise a contiguous/consecutive span of at least 8, 9, 10, 11, 12, 13,14, 15, and up to one nucleotide less than the full length nucleotidesequence of SEQ ID NOs:1-10. In a specific embodiment, the fragmentscomprise a contiguous span of at least 18 nucleotides, and up to onenucleotide less than the full length nucleotide sequence of SEQ IDNOs:1-10. Fragments should be of sufficient length to retain theirfunction according to the methods of the invention. For example,fragments of primers retain the ability to amplify their targetsequence. In specific embodiments, the fragments have one, two, three,four, or five nucleotides less than the full length nucleotide sequenceof the given polynucleotide. Such fragments may be truncated at the 5′end, the 3′ end, or both the 5′ and 3′ ends, for example. In a specificembodiment, the fragments comprise a contiguous span of at least 18nucleotides of the 5′ end of SEQ ID NOs:1-10. In another specificembodiment, the fragments comprise a contiguous span of at least 18nucleotides of the 3′ end of SEQ ID NO:1-10.

The term “molecular serotyping” as used herein is understood to meanserotyping by one of various molecular techniques rather than one ofusual immunological techniques.

An “isolated” or “purified” nucleic acid or an “isolated” or “purified”polypeptide is a nucleic acid or polypeptide that, by the hand of man,exists apart from its native environment and is therefore not a productof nature. An isolated nucleic acid or polypeptide may exist in apartially purified or substantially purified form. An isolated nucleicacid or polypeptide may also exist in a non-native environment such as,for example, a transgenic host cell. The polynucleotides of theinvention (e.g., SEQ ID NOs:1-10) are preferably in an isolated orpurified form.

The term “label” refers to any atom or molecule that may be used toprovide a detectable (preferably quantifiable) signal, and that may beattached to a nucleic acid or protein. Labels may provide signalsdetectable by fluorescence, radioactivity, colorimetry, gravimetry,X-ray diffraction or absorption, magnetism, enzymatic activity, and thelike. For example, labels such as FAM (6-carboxyfluorescein), DABCYL(4-(4′-dimethylaminophenylazo)-benzoic acid), or other flurochromes maybe utilized. For example, other flurochromes that can be added to the 5′end in place of FAM (e.g., in the beacon) include CRCG, Cy3, Cy5, OregonGreen, ROX, Texas Red, Cy 3.5, Cy 5.5, Hexachlorofluorescein, and TET.In place of DABCYL on the 3′ end as a quencher, one can use Black HoleQuencher (BHQ), for example.

In one embodiment, the polynucleotides of the subject invention comprisea nucleic acid sequence selected from the group consisting of SEQ IDNO:1-10, or a functional fragment of SEQ ID NO:1-10, wherein thepolynucleotide sequence includes a label at either the 5′ end, the 3′end, or both the 5′ and 3′ ends.

The term “nucleic acid” refers to deoxyribonucleotides orribonucleotides and polymers thereof in either single- ordouble-stranded form, composed of monomers (nucleotides) containing asugar, phosphate and a base that is either a purine or pyrimidine.Unless specifically limited, the term encompasses nucleic acidscontaining known analogs of natural nucleotides that have similarbinding properties as the reference nucleic acid and are metabolized ina manner similar to naturally occurring nucleotides. Unless otherwiseindicated, a particular nucleic acid sequence also implicitlyencompasses conservatively modified variants thereof (e.g., degeneratecodon substitutions) and complementary sequences as well as thereference sequence explicitly indicated.

The term “gene” is used to refer to any segment of nucleic acidassociated with a biological function. The term “gene” encompasses thecoding region of a protein, polypeptide, peptide or structural RNA. Theterm “gene” also includes sequences up to a distance of about 2 kb oneither end of a coding region. These sequences are referred to as“flanking” sequences or regions (these flanking sequences are located 5′or 3′ to the non-translated sequences present on the mRNA transcript).The 5′ flanking region may contain control or regulatory sequences suchas promoters and enhancers or other recognition or binding sequences forproteins that control or influence the transcription of the gene. The 3′flanking region may contain sequences that direct the termination oftranscription, post-transcriptional cleavage and polyadenylation as wellas recognition sequences for other proteins. A protein or polypeptideencoded in a gene may be full length or any portion thereof, so that allactivities or functional properties are retained, or so that onlyselected activities (e.g., enzymatic activity, ligand binding, signaltransduction, etc.) of the full-length protein or polypeptide areretained. The protein or polypeptide may include any sequences necessaryfor the production of a proprotein or precursor polypeptide. The term“gene” encompasses both cDNA and genomic forms of a coding region. Agenomic form of a coding region may be interrupted with non-codingsequences termed “introns.” The term “native gene” refers to gene thatis naturally present in the genome of an untransformed cell. The primersof the subject invention amplify at least a portion of the RNApolymerase gene of the virus to be detected within the sample.

The terms “coding region” or “coding sequence” refers to the nucleotidesequence that codes for a protein of interest or to a functional RNA ofinterest, for example antisense RNA or a nontranslated RNA. The codingregion of a protein is bounded on the 5′ side by the nucleotide triplet“ATG” that encodes the initiator methionine and on the 3′ side by one ofthe three triplets that specify stop codons (i.e., TAA, TAG, TGA). Thecoding region may be present in either a cDNA, genomic DNA or RNA form.

The terms “comprising”, “consisting of” and “consisting essentially of”are defined according to their standard meaning. The terms may besubstituted for one another throughout the instant application in orderto attach the specific meaning associated with each term.

As used in this specification and the appended claims, the singularforms “a”, “an”, and “the” include plural reference unless the contextclearly dictates otherwise. Thus, for example, a reference to “a primer”includes more than one such primer. A reference to “a polynucleotide”includes more than one such polynucleotide, and the like.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology (includingrecombinant techniques), microbiology, cell biology, biochemistry andimmunology, which are within the skill of the art. Such techniques areexplained fully in the literature, such as, “Molecular Cloning: ALaboratory Manual”, second edition (Sambrook et al., 1989);“Oligonucleotide Synthesis” (M. J. Gait, ed., 1984); “Animal CellCulture” (R. I. Freshney, ed., 1987); “Methods in Enzymology” (AcademicPress, Inc.); “Handbook of Experimental Immunology” (D. M. Weir & C. C.Blackwell, eds.); “Gene Transfer Vectors for Mammalian Cells” (J. M.Miller & M. P. Calos, eds., 1987); “Current Protocols in MolecularBiology” (F. M. Ausubel et al., eds., 1987); “PCR: The Polymerase ChainReaction”, (Mullis et al., eds., 1994); and “Current Protocols inImmunology” (J. E. Coligan et al., eds., 1991).

Following are examples that illustrate materials, methods, andprocedures for practicing the invention. The examples are illustrativeand should not be construed as limiting. Unless noted otherwise, allsolvent mixture proportions are by volume and all percentages are byweight.

EXAMPLE 1 RNA Extraction and Amplification

In a preferred embodiment, NASBA technology, in combination with aconcentration of the virus, is utilized. This step comprises filtering adesired volume of water (typically approximately 110 liters) by a methodsuch as is known in the art (Filterite filter DFN 0.45-10UN;Filterite/MEMTEC A. Corp., Timonium, Md.; Standard Methods for theExamination of Water and Wastewater, 20^(th) ed., American Public HealthAssoc., Washington, DC, 1998). Viruses are eluted with beef extract (pH9.5) and concentrated using organic flocculation. As an alternative, thewater can be filtered using vortex flow filtration (Paul, J. H. et al.Appl. Environ. Microbiol. 1991 57:2197-204. Concentration of viruses anddissolved DNA from aquatic environments by vortex flow filtration). Theviral concentrate or standard poliovirus, for example, is stored at −20°C. until extraction.

Extraction is accomplished by taking a desired amount of the concentrateor enteroviral standard (for example, poliovirus), diluted to 100 μl inDEPC Dl (diethylpyrocarbonate-treated deionized water). An exemplary kitfor accomplishing extraction comprises the Rneasy kit (Qiagen, SantaClarita, Calif.). Dilutions of the enterovirus are made toconcentrations of 9×10⁶, 9×10⁴, 9×10³, and 9×10² enteroviruses in a1.5-ml microfuge tube. Then 350 μl of RLT buffer, containing 10 μlβ-mercaptoethanol per 1.0 ml RLT buffer, is added to the tubes, and thetubes are capped and placed in a 95° C. water bath for 10 minutes,followed by placement in an ice bath for 5 minutes. An amount ofethanol, here 250 μl, is added and mixed well by pipetting. This mixture(typically 700 μl), including any precipitate, is added to the spincolumn, and the tube is placed in a 2-ml collection tube, which isplaced in a microfuge for 15 seconds at ≧10,000 rpm. 700 μl of bufferRW1 is pipetted into the column and microfuged for 15 seconds at ≧10,000rpm to wash. Then 500 μl of RPE buffer is pipetted into the tube, usinga new collection 2.0-ml tube, and microfuged for 15 seconds at ≧10,000rpm. 500 μl RPE is pipetted onto the column and centrifuged for 2minutes at maximum speed to dry the column using the same collectiontube. The mixture is transferred to a new 1.5-ml collection tube, with30 μl Rnase-free water pipetted, and the tube is spun for 1 minute toelute. Then 1 U (unit) of Rnasin (PROMEGA) per microliter of sample isadded.

Amplification of the enterovirus (here, for example, poliovirus) ispreferably accomplished with the use of a kit (BIOMERIEUX (Durham,N.C.)), using two primer sequences, EntP1 (=JP127) and EntP2 (=JP128).ENTP1 comprises5′-AAT-TCT-AAT-ACG-ACT-CAC-TAT-AGG-GAG-AAG-GAC-CGG-ATG-GCC-AAT-CCA-A-3′(SEQID NO:1); EntP2 comprises 5′-CCT-CCG-GCC-CCT-GAA-TGC-GGC-TAA-3′(SEQ IDNO:2). The primers are gel purified. Lyophilized primers are taken up insterile DEPC-D1 to a final concentration of 100 μM, and aliquotted. Thealiquotted samples can be utilized immediately by diluting to 10 μM, orthey can be frozen for future use. If frozen prior to use, they arethawed and diluted to 10 μM.

A clean workbench should preferably be set up with UV-sterilization andhot blocks set at 65° C. and 41° C. Using a kit such as the BIOMEREIEUXkit, add 50 μl accusphere diluent to lyophilized accusphere, and vortexwell. Then 5 μl of each diluted (10 μM) EntP1 and EntP2 primers areadded to 50 μl dissolved accusphere for a total of 60 μl, which issufficient for 11-12 reactions.

80 mM KCl is prepared. For example, 80 mM KCl can be prepared utilizingthe NASBA kit by adding 16 μl NASBA KCl and 14 μl NASBA water. The 90 μlprimer/accusphere mix is combined with the 30 μl KCl. For a positivecontrol, the control contained in the kit can be used. In this case, 50μl NASBA water plus the lyophilized NASBA control are added.

The reaction is set up by adding 5 μl Dl+10 μl of primer/KCl mixture toa sterile 1.5-ml microfuge tube, which serves as a blank. For apoliovirus or unknown sample, 5 μl Dl+10 μl primer/KCl mixture is addedto a sterile 1.5 ml microfuge tube. For a kit control, aliquot out 15 μlof the positive control mixture to a sterile 1.5 ml microfuge tube.

The tubes are placed in the 65° C. hot block for 5 minutes and in a 41°C. hot block for 5 minutes, and 5 μl of the NASBA enzyme mixture isadded, with mixing accomplished preferably by flicking, not trituration.For most assays, this “hot start” is not necessary, however. The tubesare incubated in the 41° C. hot block for 5 minutes, then pulse spun ina microfuge for 1-2 sec, incubated for 90 minutes in the 41° C. hotblock, removed from the hot block, and immediately utilized fordetection. Alternatively, if the samples are not used immediately, theycan be frozen at −80° C. immediately and stored.

It should be noted that utmost care should be taken to keep extractionareas separate from amplification areas. At all times, positive controlsshould be kept separated from other samples. For example, the positivecontrols should never be in the same rack as negatives or unknowns. Tofurther decrease the chance of cross-contamination, aerosol pipette tipsshould be used, gloves worn, and gloves changed frequently.

Detection is accomplished using, for example, a 7% acrylamide gel, runfor 3 to 5 hours, followed by ethidium bromide staining. Alternatively,dot blotting and probing can be used. A list of preferred probes isprovided in Table 1 below. One probe useful for poliovirus comprises5′-TAC-TTT-GGG-TGT-CCG-TGT-TTC-3′(SEQ ID NO:3). The probes can belabeled, such as by using a TROPIX, Inc. (Bedford, Mass.), Southern StarChemiluminesent Detection System, version A.2. Detection of specificenteroviral types can be accomplished by probing with specificviral-type oligonucleotide probes.

EXAMPLE 2 Methods to Detect and Quantitate Enteroviruses and Norovirusesby Real Time Nucleic Acid Sequence Based Amplification (NASBA)

This method has been developed to detect enteroviruses and genogroup IInoroviruses from environmental samples. Environmental samples can befresh or saline waters, surfaces, and stool samples. The methods shouldalso be appropriate for clinical samples (blood, urine, cerebrospinalfluid, etc.)

A. Extraction of Viral RNA from Aqueous Samples

If samples are suspected to have high concentrations of viruses (e.g.,raw sewage, wastewater, septage), then there is no need to concentrate.For concentration from seawater, a modified method of Katayama, H. etal. (Appl. Environ. Microbiol., 2002, 68(3):1033-1039) is recommended.

B. Collection of Enteroviruses and Noroviruses from Seawater

-   1. Collect 500-1000 ml of the seawater to be tested (if larger    volumes are to be sampled, increase the diameter of the filter and    the filtration manifold to be used).-   2. Using a sterile 47 mm filtration unit fitted with a sterile    Millipore HA filter, filter the water sample using mild vacuum    (10-15 psi).-   3. When the filtration is complete, wash the filter by filtering    through 200 ml of 0.5 mM H₂SO₄ to remove cations. This wash is    discarded.-   4. The filtration unit is connected to a small collection vacuum    flask. 2.0 ml of 1 mM NaOH is added and filtered through the filter.    The filtrate is neutralized with 50 mM H₂SO₄ and used in the RNA    extraction protocol below.

C. Collection of Noroviruses from Stool Samples

-   1. Make sure all protocols are performed in a Biological Safety    Cabinet.-   2. Bring 100 to 500 ul of feces to 1.0 ml by adding sterile,    RNase-free Phosphate Buffered saline (PBS).-   3. It is convenient to divide this into two 500 ul aliquots in    sterile 2 ml microcentrifuge tubes. Add 1.5 ml freon (1,12    trichlorotrifluoroethane) and mix well by vortexing.-   4. Spin at 5000×G for 10 min in a microcentrifuge. Collect the upper    aqueous phase, being sure not to transfer particulate matter. If the    supernatant is clear, proceed to RNA extraction. If not, perform a    second freon extraction.-   5. Collect the supernatant and proceed to the RNA extraction    protocol.

D. RNA Extraction Protocol

This protocol uses the Qiagen RNeasy Kit and protocol (RNeasy Mini KitQiagen Corporation #74106), whereas other RNA extraction protocols maybe used.

-   1. 100 ul of the extract from stool samples or environmental samples    as described above is added to 350 ul of the RLT buffer (containing    β-mercaptoethanol) in the Qiagen RNeasy kit and briefly vortexed.-   2. This mix is incubated at room temperature for 10 minutes.-   3. 250 μl of 100% EtOH is added, and the entire 700 μl mixture    placed onto an RNeasy column.-   4. The column is centrifuged at 10,000 rpm for 30 seconds.-   5. The flow through is discarded.-   6. 700 μl of Buffer RW1 (from the RNeasy kit) is added to the column    and the column centrifuged at 10,000 rpm for 30 seconds.-   7. The collection tube is discarded and 500 μl Buffer RPE (+EtOH;    RNeasy Kit) is added to the column.-   8. The column is centrifuged at 10,000 rpm for 30 seconds and the    flow through discarded.-   9. Another 500 μl RPE is added to the column and the column    centrifuged for 2 minutes at 10,000 rpm.-   10. The collection tube is discarded and the column centrifuged    again at full speed for 1-2 minutes.-   11. The column is placed in a 1.5 ml collection tube and 50 μl of    RNase free water is added directly to the column. The column is    centrifuged at full speed for 1 minute.-   12. RNA (2.5 μl) is then directly used for NASBA or diluted (1:10,    1:100) to dilute potential inhibitors.

E. NASBA Detection and Quantitation of Noroviruses

Instrumentation: Specialized instrumentation developed for Q-PCR(quantitative PCR) that enables incubation of samples at a controlledtemperature while simultaneously measuring fluorescence is required.This includes but is not limited to the ABI Model 7700 SequenceDetection System, or the BioMerieux EasyQ reader (the latter only worksfor NASBA).

NASBA Regents: NASBA Reagents should include the BioMerieux NuclisensBasic Kit (#285053)

NASBA Primers and Beacon: Primers and beacons can be synthesized by amanufacturer of oligonucleotides (e.g., Qiagen-Operon) and should,preferably, be gel-purified. Tables 2 and 3 list the sequences of theNASBA primers and Molecular Beacon for Enteroviruses and Noroviruses. Itis recommend that the primers and beacon be dissolved in sterileRNase-free water at a stock concentration of 100 uM and aliquoted intosmall volumes and frozen at −80° C. TABLE 1 Primers and Beacon for theamplification of the enterovirus 5′ UTR (untranslated region) PrimerSequence Ent P1 5′AATTCTAATACGACTCACTATAGGGAGAAGGACCG GATGGCCAATCCAA-3′(SEQ ID NO:1) Ent P3 5′-GGTGTCCGTGTTTCCTTTTA-3′ (SEQ ID NO:4)EnteroBeacon 5′FAM-CATGCGTGGCTGCTTATGGTGACAATCGCAT G-DABCYL-3′ (SEQ IDNO:5)These primers will amplify a 106-108 base pair (bp) amplicon.

TABLE 2 Primers and Beacon for the amplification of the norovirus RNApolymerase gene. Primer Sequence Nor3 5′CAATGGAATTCCATCGCCCA 3′ (SEQ IDNO:6) Nor4 5′AATTCTAATACGACTCACTATAGGG-AGAA- GTTGTCACGATCTCATCATCA 3′(SEQ ID NO:7) NorBeacon 2 5′-FAM-CATCGGACATCATACAGGCTAATTCCGATG-DABCYL-3′ (SEQ ID NO:8)FAM is 6-carboxyfluorescein, and DABCYL is4-(4′-dimethylaminophenylazo)-benzoic acid. Other flurochromes that canbe added to the 5′ end in place of FAM in the beacon include, forexample, CRCG, Cy3, Cy5, Oregon Green, ROX, Texas Red, Cy 3.5, Cy 5.5,Hexachlorofluorescein, and TET. In place of DABCYL on the 3′ end as aquencher, one can use Black Hole Quencher (BHQ), for example.

These primers will amplify a 123 bp fragment of the Genogroup II RNApolymerase gene of many Noroviruses. The T7 Promoter region isunderlined.

NASBA Protocol

-   1. Dilute primer stocks 1:4 (2 μl primer to 6 μl H₂O).-   2. Dilute beacon stock 1:4 (1.5 μl beacon to 4.5 μl H₂O).-   3. Dilute stock 12 mM ROX (5-[and 6]-carboxy-X-rhodamine) 1:100 (1    μl ROX to 99 μl H₂O).-   4. Primer Mix: 4 μl each primer (25 μM), 1 μl Beacon (25 μM) and 1    μl ROX (120 μM) results in each primer becoming 10 μM, Beacon 2.5 μM    and the ROX 12 μM.-   5. Dilute the KCl (yellow capped tube in BioMerieux Nuclisens Basic    Kit) 16 μl KCl to 14 μl H2O-   6. Dilute the reagent sphere (Blue capped tube in BioMerieux    Nuclisens Basic Kit) in 80 μl of diluent and vortex to mix well.-   7. Add the entire 30 μl KCl mix to the reagent mix (Final    Concentration 80 μM KCl).-   8. Add the entire 10 μl primer mix (Concentrations at this point are    800 nM primers, 200 nM beacon, and 1 μM ROX).-   9. Place 5 μl of this mix into 24 tubes 200 μl optical PCR tubes    (such as Applied Biosystems Optical Tubes, part #4316567) (Final    concentrations are 400 nM primers, 100 nM beacon and 500 nM ROX).-   10. Add 55 μl of enzyme diluent to the enzyme sphere (Red capped    tube in the BioMerieux Basic Kit) (start the timer for 20 min).-   11. Add 2.5 μl of RNA extract (see above) or RNase-free DI water    (blank) to each reaction tube.-   12. After the 20 minutes (enzyme sphere dissolution time), start the    precycle which is 2 minutes at 65° C. and then 41° C. for 2 minutes    in the ABI 7700 or equivalent instrument.-   13. During the precycle, aliquot 2.5 μl of enzyme into a strip of    caps of optical tubes (such as Applied Biosystems Optical Caps, part    no. 4323032).-   14. Add enzyme to the tubes by spinning from the caps.-   15. Start NASBA for 41° C. for 90 min in the ABI 7700 or equivalent    instrument.-   16. Running a series of standards or dilutions of a known positive    stool sample is recommended. Alternatively, one can make transcript    in vitro from cloning a portion of the RNA polymerase gene.-   17. When the cycle is complete, analyze the data, set an appropriate    threshold fluorescence, and relate the standards to the time to    positivity (time at which samples display a significant increase in    fluorescence). Plot the time to positivity vs. the log concentration    of the standard. Use this regression to calculate the approximate    viral load (or RNA concentration) in unknown samples.

Results obtained with the Method

Standard curves were prepared with Poliovirus S1 that were counted byepifluorescence microscopy. A typical standard curve appears in FIG. 2B.TABLE 3 Specificity of amplification using enteroviruses EnterovirusSource # of particles Amplification Coxsackie B4 T. Scott 2.2 × 10⁴ +Coxsackie B5 T. Scott 1.55 × 10⁵  + Echovirus 1 T. Scott 2.32 × 10⁵  +Echovirus 4 T. Scott 9.19 × 10⁴  + Echovirus 5 T. Scott 1.07 × 10⁵  +Echovirus 7 T. Scott 2.53 × 10⁵  + Echovirus 14 T. Scott 1.91 × 10⁵  +Echovirus 26 T. Scott 6.69 × 10⁴  + Poliovirus 1 T. Scott 5.6 × 10⁴ +Coxsackie B1 ATCC VR-28 6.5 × 10⁴ + Coxsackie B2 ATCC VR-29 3.0 × 10⁴ +Coxsackie B3 ATCC VR-30 5.4 × 10⁴ + Coxsackie A9 ATCC VR-186 3.7 × 10⁴ +Coxsackie A16 ATCC VR-174 5.5 × 10⁴ + Enterovirus 70 ATCC VR-836 1.6 ×10⁵ + Enterovirus 71 ATCC VR-784 3.6 × 10⁴ +

Table 3 shows the specificity of the primers toward enteroviral targets.Sixteen of 16 enteroviruses were detected by the method. Table 4 showsthe specificity or lack of reactivity toward non-enteroviral targets. Nonon-enteroviral targets tested amplified using the EntP1/EntP3 primersets. TABLE 4 Negative viruses tested by the Enterovirus Real Time NASBAassay. Name Strain Source Amplification (−) Control USF-DOH − NorovirusG2 G2 USF-DOH − Herpesvirus USF-DOH − Influenza A (H1N1) A USF-DOH −Influenza B B USF-DOH − Rotavirus USF-DOH − Adenovirus USF-DOH −Rhinovirus USF-DOH − Hepatitis A USF-DOH − Reovirus USF-DOH − DengueUSF-DOH −

Current detection methods for norovirus do not permit quantification ofvirus. To overcome this limitation, the present inventors have taken anapproach in which a standard curve is produced based on known transcriptconcentrations from which unknown samples can then be extrapolated todetermine virus concentrations. This method assumes that each viralparticle renders one copy of the target sequence. To generate atranscript standard, primers were designed to specifically amplify thetarget region amplified in the NASBA reaction plus approximately 100bases of the flanking RNA both up and downstream. Reverse transcriptionPCR with these primers resulted in a 350 base pair product that wassubsequently cloned into pCRII (INVITROGEN Corporation, Carlsbad,Calif.). Using an internal SP6 promoter region on the vector, in vitrotranscript of the Norovirus target region was generated and quantified.

Standard curves using serially diluted transcript from 1 pg to 0.01 fgwere generated. R² values were typically around 0.9 (FIGS. 2A and 2B).The NASBA assay could routinely detect 1 fg transcript (approx. 10⁴viral particles). Furthermore, 0.1 fg was detected in most runs (75%).The present inventors have compared these detection limits to thoseobtained using a side-by-side real time PCR (Taqman) assay for norovirusdetection and have determined that the sensitivity of each assay issimilar.

Noroviruses are a genetically diverse group of viruses. This sequencedivergence has somewhat hampered the progress of the development ofreliable nucleic acid sequence based detection methods. To evaluate thismethod's ability to overcome this issue, the present inventors extractedvirus from 114 contaminated fecal samples. Raw stool samples collectedfrom outbreak patients in Florida over the past four years wereFreon-extracted to remove most contaminants. RNA was then extracted fromthe viruses using the RNeasy extraction protocol (QIAGEN Corporation,Valencia, Calif.). To date, forty-five of these samples have beenevaluated using the method of the invention. In these tests, 78% of thesamples tested positive for norovirus by NASBA. Sequence analysis onfive of the negative samples revealed that the beacon region wassignificantly different from the probe sequence. The other negativesamples are currently being evaluated in the same manner.

EXAMPLE 3 Quantitative Detection of Enteroviruses by Real Time NASBAusing an internal control (IC-NASBA)

Because of the three enzymes involved in NASBA, each with theirdifferent kinetics, the time to positivity (TTP) approach taken inQuantitative (Real-Time) PCR does not provide ideal standard curves withNASBA. Data sets typically have reaction kinetics that do not easilylend themselves to a simple threshold cycle (Ct) analysis. This resultsin standard curves with lower coefficients of determination (r²=0.7-0.8)for NASBA compared to those for Real-Time PCR (typically >0.95).

The present inventors have designed a specific internal controlcontaining a different beacon hybridization site. Thus, the sameamplification primers are used for both the calibrator and the targetmRNA. However, a second molecular beacon containing a different fluor(6-ROX) is used, which hybridizes to the unique internal site in thecalibrator molecule (FIG. 4). The amplification of both the calibratorand the target are measured simultaneously by measuring fluorescence attwo differing wavelengths (thus the need for two fluorescence channelsin the sensor). The present inventors have made a synthetic RNA internalcontrol for enteroviruses that contains the beacon site for an algal RNA(as shown below).

A. Methods for IC-NASBA

Enteroviral Internal control: (SEQ ID NO:9)5′GGTGTCCGTGTTTCCTTTTATTTTTATTGCTTAGTCTCGGGTTATTTTTTCACAGATTGTTATCATAAAGCGAATTGGATTGGCCATCCGGT-3′

Enteroviral Internal control Beacon: (SEQ ID NO:10)5′-ROX-CGATCGCTTAGTCTCGGGTTATTTTTTCGATCG-DABCYL-3′

To generate the internal control molecule, two oligonucleotides weredesigned that spanned the entire calibrator sequence and contained a 20base overlap with each other. The oligos (serving as both template andprimers) were placed in a PCR reaction (100 pmols each) and run understandard PCR conditions with an annealing step of 55° C. for twentycycles. Reaction products were visualized on a 1% agarose gel stainedwith Ethidium Bromide. Amplicons were TA TOPO cloned into the pCRIIcloning vector according to the manufacturer's instructions (InvitrogenCorporation, Carlsbad, Calif.). Clones were checked for directionalityby PCR screening using an internal upstream M13 reverse primer and theassay's downstream (T7) primer. Two clones appearing to have the insertin the correct orientation were grown overnight in LB supplemented with50 μg/ml of Kanomycin and the plasmid was extracted using the WizardMidi-plasmid purification kit (PROMEGA Corporation, Madison, Wis.). Theplasmid was sequenced using the internal vector M13 reverse primer toinsure the insert's integrity. The plasmid was then linearized bydigestion with NotI, the enzyme removed using the Wizard DNA clean-upkit (PROMEGA Corporation, Madison, Wis.), and run-off transcriptsgenerated from an upstream SP6 promoter using the Riboprobe in vitrotranscription kit (PROMEGA Corporation, Madison, Wis.). RNA wasquantified and stored as described earlier.

B. Primer Mix and Beacons

-   1. Primers (e.g., ENTP1 and ENTP3; SEQ ID NOs:1 and 4, respectively)    are diluted for final concentrations of 400 nM.-   2. Both beacons (e.g., SEQ ID NOs:5 and 10)are diluted for final    concentrations of 100 nM.-   3. Internal control dilution is dependent on specific assay—for    enteroviruses, addition of 10⁴ copies/reaction is suitable.-   4. 4 μl of each primer and 1 μl of each beacon are added to the    primer mix (no 6-Rox is added).-   5. 1 μl diluted internal control is added to the primer mix for a    total volume of 11 μl.

C. Reagent Mix

16 μl KCl stock solution is mixed with 13 μl NASBA water. The wholevolumes of primer mix (11 μl) and KCl solution (29 μl) are added to the80 μl reagent mix for a final volume of 120 μl, the same as if thebioMerieux protocol is followed.

-   1. Add the entire 10 μl primer mix (concentrations at this point are    400 nM primers, 100 nM beacon).-   2. Place 5 μl of this mix into 24 tubes 200 ul optical PCR tubes    (such as Applied Biosystems Optical Tubes, part #4316567).-   3. Add 55 μl of enzyme diluent to the enzyme sphere (Red capped tube    in the BioMerieux Basic Kit) (start the timer for 20 minutes).-   4. Add 2.5 μl of RNA extract (see above) or RNase-free DI water    (blank) to each reaction tube.-   5. After the 20 min (enzyme sphere dissolution time) start the    precycle which is 2 minutes at 65° C. and then 41° C. for 2 min in    the ABI 7700 or equivalent instrument.-   6. During the precycle, aliquot 2.5 μl of enzyme into a strip of    caps of optical tubes (such as Applied Biosystems Optical Caps, part    no. 4323032).-   7. Add enzyme to the tubes by spinning from the caps.-   8. Start NASBA for 41° C. for 90 min in the ABI 7700 or equivalent    instrument.-   9. It is recommended to run a standard curve using extractions of    log dilutions of the enterovirus, usually from 10⁴ to 10² viruses    calculated to be in the 2.5 μl RNA extract.

D. Results

FIG. 5 shows the results of a typical IC-NASBA reaction containing10,000 Internal Control copies. The ratio of the Ct of known standardsto the Ct of the internal control is plotted against the standardconcentration. This ratio is linearly correlated with concentration.Thus, once this relationship is established, it is used to determine theconcentration of unknowns. FIGS. 6A and 6B show standard curves ofenteroviral RNA signals plotted against the Ct (FIG. 6A) and against theCt ratio (FIG. 6B). A much better relationship is apparent in the Ctratio method (R²=0.9882 for Ct ratio [IC-NASBA] vs. 0.7379 for Ct valuemethod.

All patents, patent applications, provisional applications, andpublications referred to or cited herein are incorporated by referencein their entirety, including all figures, tables, and sequences to theextent they are not inconsistent with the explicit teachings of thisspecification.

It should be understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication.

1. An isolated polynucleotide selected from the group consisting of SEQID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ IDNO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10, or afragment of any of the foregoing.
 2. The isolated polynucleotide ofclaim 1, which is SEQ ID NO:1.
 3. The isolated polynucleotide of claim1, which is SEQ ID NO:2.
 4. The isolated polynucleotide of claim 1,which is SEQ ID NO:3.
 5. The isolated polynucleotide of claim 1, whichis SEQ ID NO:4.
 6. The isolated polynucleotide of claim 1, which is SEQID NO:5.
 7. The isolated polynucleotide of claim 1, which is SEQ IDNO:6.
 8. The isolated polynucleotide of claim 1, which is SEQ ID NO:7.9. The isolated polynucleotide of claim 1, which is SEQ ID NO:8.
 10. Theisolated polynucleotide of claim 1, which is SEQ ID NO:9.
 11. Theisolated polynucleotide of claim 1, which is SEQ ID NO:10.
 12. A methodfor detecting the presence of an Enterovirus in a sample, comprisingcontacting a polynucleotide region of the 5′-untranslated region of theEnterovirus genome with amplification primers; amplifying thepolynucleotide region of the 5′-untranslated region; and detecting thepresence of an amplified polynucleotide, wherein the presence of theamplified product is indicative of the presence of the Enterovirus inthe sample.
 13. The method of claim 12, wherein the amplificationprimers are selected from the group consisting of SEQ ID NO:1, SEQ IDNO:2, and SEQ ID NO:4, or a fragment of any of the foregoing.
 14. Themethod of claim 12, wherein said method further comprises contacting thepolynucleotide region of the 5′-untranslated region with a labeledmolecular beacon.
 15. The method of claim 14, wherein the labeledmolecular beacon comprises SEQ ID NO:5.
 16. The method of claim 12,wherein said amplifying comprises nucleic acid sequence basedamplification (NASBA).
 17. The method of claim 12, wherein saidamplifying comprises RT-PCR.
 18. The method of claim 12, wherein saiddetecting comprises hybridization of the amplified product with a probe.19. The method of claim 18, wherein said detecting comprising serotypingthe Enterovirus with a probe specific for an Enterovirus serotype. 20.The method of claim 12, wherein the sample is a biological sample. 21.The method of claim 12, wherein the sample is an environmental sample.22. The method of claim 12, wherein the Enterovirus is selected from thegroup consisting of Polio 1, 2, 3; Coxsackie A 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24; CoxsackieB 1, 2, 3, 4, 5, 6; ECHO 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34; Entero 68, 69, 70, 71; and Entero 72 (Hepatitis A).
 23. Themethod of claim 12, wherein said method further comprises determiningthe quantity of Enterovirus within the sample.
 24. A method fordetecting the presence of a Norovirus in a sample, comprising contactinga polynucleotide region of the RNA polymerase gene of the Norovirus withamplification primers; amplifying the polynucleotide region of the RNApolymerase gene; and detecting the presence of an amplifiedpolynucleotide, wherein the presence of the amplified product isindicative of the presence of the Norovirus in the sample.
 25. Themethod of claim 24, wherein the amplification primers are SEQ ID NO:6and SEQ ID NO:7, or a fragment of any of the foregoing.
 26. The methodof claim 24, wherein said method further comprises contacting thepolynucleotide region of the RNA polymerase gene with a labeledmolecular beacon.
 27. The method of claim 26, wherein the labeledmolecular beacon comprises SEQ ID NO:8.
 28. The method of claim 24,wherein said amplifying comprises nucleic acid sequence basedamplification (NASBA).
 29. The method of claim 24, wherein saidamplifying comprises RT-PCR.
 30. The method of claim 24, wherein saiddetecting comprises hybridization of the amplified product with a probe.31. The method of claim 30, wherein said detecting comprising serotypingthe Norovirus with a probe specific for a Norovirus serotype.
 32. Themethod of claim 24, wherein the sample is a biological sample.
 33. Themethod of claim 24, wherein the sample is an environmental sample. 34.The method of claim 24, wherein the Norovirus is a genotype II strainselected from the group consisting of GII-1 (Hawaii virus), GII-2(Melksham virus), GII-3 (Mexico virus), and GII-4 (Grimsby virus). 35.The method of claim 24, wherein said method further comprisesdetermining the quantity of Norovirus within the sample.
 36. A kit fordetecting the presence of enterovirus and/or norovirus in a sample,comprising two or more isolated polynucleotides selected from the groupconsisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ IDNO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10, or afragment of any of the foregoing.